Wohlin P, Zeisig M, Gustafsson J A, Möller L
Department of Bioscience, Karolinska Institute, Huddinge, Sweden.
Chem Res Toxicol. 1996 Sep;9(6):1050-6. doi: 10.1021/tx960050a.
DNA adducts of 2-amino-1 methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-diMeIQx), synthesized in vitro with calf thymus DNA and formed in vivo in the male Wistar rat, were enriched from digested DNA by butanol extraction before 32P-postlabeling. The recovery after butanol enrichment was 79% and 32% for in vitro modified PhIP- and 4,8-diMeIQx-DNA adducts, respectively. Crude postlabeling mixtures were chromatographically separated by high-performance liquid chromatography with on-line 32P-detection (32P-HPLC). The major PhIP- and 4,8-diMeIQx-DNA adducts formed in vitro cochromatographed with the respective pdGp-C8 adduct standard. 32P-HPLC was also used to separate hydrolysates of in vitro formed PhIP-DNA and 4,8-diMeIQx-DNA that had been 32P-postlabeled under ATP-deficient conditions. The adduct recovery of the ATP-deficient method relative to the improved butanol enrichment procedure was 29% and 59% for total PhIP-DNA and 4,8-diMeIQx-DNA adducts, respectively. Simplified DNA adduct patterns were obtained when the postlabeling mixtures were incubated with nuclease P1, suggesting incomplete DNA hydrolysis. After nuclease P1 treatment, the major DNA adducts of PhIP and 4,8-diMeIQx formed in vitro cochromatographed with the respective pdG-C8 adduct standard. In vivo PhIP formed what appeared to be multiple DNA adducts; however, after nuclease P1 treatment the PhIP-associated peaks were concentrated into a single peak cochromatographing with pdG-C8-PhIP, 4,8-diMeIQx formed multiple DNA adducts in vivo. Nuclease P1 treatment resulted in two 4,8-diMeIQx related peaks, one cochromatographing with pdG-C8-4,8-diMeIQx. The second peak remains unidentified. The improved workup procedures in combination with the high resolution and reproducibility of the 32P-HPLC system should be useful for characterization of PhIP- and 4,8-diMeIQx-DNA adducts in DNA modified by complex mixtures.
2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶（PhIP）和2-氨基-3,4,8-三甲基-3H-咪唑并[4,5-f]喹喔啉（4,8-二甲基-IQx）的DNA加合物，在体外与小牛胸腺DNA合成，并在雄性Wistar大鼠体内形成，在32P后标记之前通过丁醇萃取从消化的DNA中富集。丁醇富集后，体外修饰的PhIP-DNA和4,8-二甲基-IQx-DNA加合物的回收率分别为79%和32%。粗制的后标记混合物通过在线32P检测的高效液相色谱（32P-HPLC）进行色谱分离。体外形成的主要PhIP-DNA和4,8-二甲基-IQx-DNA加合物与各自的pdGp-C8加合物标准品共色谱。32P-HPLC还用于分离在ATP缺乏条件下进行32P后标记的体外形成的PhIP-DNA和4,8-二甲基-IQx-DNA的水解产物。相对于改进的丁醇富集程序，ATP缺乏方法对总PhIP-DNA和4,8-二甲基-IQx-DNA加合物的加合物回收率分别为29%和59%。当后标记混合物与核酸酶P1孵育时，获得了简化的DNA加合物图谱，这表明DNA水解不完全。核酸酶P1处理后，体外形成的PhIP和4,8-二甲基-IQx的主要DNA加合物与各自的pdG-C8加合物标准品共色谱。体内PhIP形成了似乎是多个DNA加合物；然而，核酸酶P1处理后，与PhIP相关的峰集中到一个与pdG-C8-PhIP共色谱的单峰中，4,8-二甲基-IQx在体内形成多个DNA加合物。核酸酶P1处理产生了两个与4,8-二甲基-IQx相关的峰，一个与pdG-C8-4,8-二甲基-IQx共色谱。第二个峰仍未鉴定。改进的后处理程序与32P-HPLC系统的高分辨率和重现性相结合，应该有助于表征复杂混合物修饰的DNA中的PhIP-和4,8-二甲基-IQx-DNA加合物。
